Sofie Lust

Identification of an Ire1alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma.

Activation of the adaptive Ire1-XBP1 pathway has been identified in many solid tumors and hematologic malignancies, including multiple myeloma (MM). Here, we report the identification of STF-083010, a novel small-molecule inhibitor of Ire1. STF-083010 inhibited Ire1 endonuclease activity, without affecting its kinase activity, after endoplasmic reticulum stress both in vitro and in vivo. Treatment with STF-083010 showed significant antimyeloma activity in model human MM xenografts. Similarly, STF-083010 was preferentially toxic to freshly isolated human CD138(+) MM cells compared with other similarly isolated cell populations. The identification of this novel Ire1 inhibitor supports the hypothesis that the Ire1-XBP1 axis is a promising target for anticancer therapy, especially in the context of MM.

Steroid effects on ZAP-70 and SYK in relation to apoptosis in poor prognosis chronic lymphocytic leukemia

There is resurgent interest in glucocorticoids (GCs) in the treatment of poor prognosis chronic lymphocytic leukemia (CLL). Little is known however on how GCs induce apoptosis in CLL. Methylprednisolone (MP) induces apoptosis in ZAP-70 positive CLL more readily than in ZAP-70 negative CLL, which is in contrast to the effects of radiation and chemotherapy. The increased GC sensitivity of ZAP-70+ CLL was studied in relation to the expression status of ZAP-70 and the related signal transducing tyrosine kinase SYK. Both ZAP-70 and SYK were downregulated by GC treatment. Moreover, SYK was dephosphorylated by the phosphatase PTP1B of which the expression and translation levels were induced by GCs. Inhibition of PTP1B successfully restored ZAP-70 expression and SYK phosphorylation but did not interfere with GC-induced apoptosis. Therefore, the downregulation of ZAP-70 and P-SYK per se during treatment with GCs is not sufficient to induce apoptosis, and different mechanisms must therefore be responsible for the increased steroid sensitivity of ZAP-70+ CLL.

The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562.

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

ER stress in diffuse large B cell lymphoma: GRP94 is a possible biomarker in germinal center versus activated B-cell type

The process of B-cell development is characterized by the activation of the unfolded protein response. Under certain circumstances, the unfolded protein response can be manipulated in a cell death-inducing way. Therefore, tackling the unfolded protein response might be an attractive strategy in the treatment of diffuse large B-cell lymphomas. Our research showed more basal unfolded protein response activity and differences in the inducibility of ER stress in activated B-cell versus germinal center cell lines. Moreover, the diffuse large B cell lymphoma patient data revealed that the glucose-regulated protein 94 is new potential discriminator for diffuse large B cell lymphoma.

Lipoprotein lipase SNPs rs13702 and rs301 correlate with clinical outcome in chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by a heterogeneous clinical course. This variability in clinical course has spiked the search for prognostic markers able to predict patient evolution at the moment of diagnosis. Markers demonstrated to be of value are the mutation status of the immunoglobulin heavy chain variable region genes (IGHV) and lipoprotein lipase (LPL) expression. High LPL mRNA expression has been associated with short treatment free (TFS) and decreased overall survival (OS) in CLL. The LPL SNPs rs301 (T<C), rs328 (C<G) and rs13702 (T<C) have been associated with various metabolic disorders, but the association with CLL evolution is unknown. Here, in a cohort of 248 patients, we show that patients with the LPL SNP rs13702 wild-type T/T genotype had significantly shorter OS than patients with C/C and T/C genotypes (median time until CLL related death: 90 and 156 months respectively, p=0.008). The same was observed for LPL SNP rs301 (median time until CLL related death T/T: 102 and C/C, T/C: 144 months, p=0.03). Both SNPs rs301 and rs13702 were significantly associated with each other and notably, no association was found between IGHV status and presence of the SNP genotypes, indicating that these LPL SNPs are reliable prognostic markers that could add extra prognostic and predictive information to classical markers and help to improve the management of CLL.

Mimicking the tumour microenvironment of chronic lymphocytic leukaemia in vitro critically depends on the type of B-cell receptor stimulation

Background:The B-cell receptor (BCR) has a key role in the cross-talk between chronic lymphocytic leukaemia (CLL) cells and the tissue microenvironment, which favours disease progression by promoting proliferation and drug resistance. In vitro studies on downstream signalling and functional effects of CLL BCR ligation often report contradictory results, in part owing to the lack of a standardised stimulation protocol. Our aim was to define a biologically relevant and robust in vitro stimulation method with regard to cellular phenotypic and transcriptional responses.Methods:We evaluated mRNA (FOS, MYC, LPL) and protein (CD54, CD19, CD62L, CD184) expression of genes modulated by BCR triggering in immunoglobulin heavy-chain variable region genes (IGHV)-mutated and -unmutated CLL cells, after stimulation using soluble or immobilised anti-IgM antibodies from different suppliers.Results:The effect of BCR stimulation on gene and protein expression was comparable in all CLL patients, irrespective of IGHV mutation status. However, immobilised anti-IgM stimulation elicited clear and robust changes in gene and protein expression, whereas the response to soluble anti-IgM was far less obvious.Conclusions:These data indicate that the method of BCR stimulation is of major importance regarding responsiveness of CLL cells in the context of the tumour microenvironment, whereas genetic differences in the BCR pathway are less critical.

86 – The Anti-invasive and Proapoptotic Effect of Xanthohumol: Potential Use in Cancer

Abstract In recent years, a lot of attention has been paid to phytochemicals such as flavonoids with anticancer effects since they may interact with cellular signaling pathways controlling proliferation, differentiation, apoptosis and invasion. Invasion is the hallmark of malignancy, and the search for anti-invasive agents remains a challenge. A number of studies reported a promising role for xanthohumol (X) as a chemopreventive agent, since it can modulate the carcinogen metabolism and act by cytotoxic/-static mechanisms. Recently, X was investigated for its anti-invasive activity on human breast cancer cell lines and shown to inhibit the invasion of breast cancer cells in different invasion assays. One of the possible mechanisms of the anti-invasive effect of X includes the involvement of the E-cadherin/catenin invasion-suppressor complex. Another mechanism by which X influences the invasive behavior is by reducing the number of invasive cells (i.e. by promoting cell death). Recent data demonstrate that X induces a specific stress reaction (i.e. endoplasmic reticulum stress) followed by the unfolded protein response and programmed cell death (apoptosis). In this overview, we highlight the effect of X on invasion and survival of breast cancer cells and the mechanisms associated with these effects.